Plant breeding programs require analyses of phenotypes for a large number of plants. These analyses involve measurement of a wide array of plant characteristics including plant morphology, disease and environmental stress tolerance, seed quality, and yield. In addition to the evaluation of phenotype, breeding programs often necessitate determination of genotype, for example, to identify a DNA marker associated with a specific phenotype or to confirm the presence of a transgene in a transgenic plant. Therefore extraction of plant genomic DNA may also be necessary.
Traditionally, analysis of phenotype in a breeding program has been conducted through visual evaluation and manual measurement of morphological characteristics. However, due to the large number of plants that must be evaluated and to the small differences when plants are evaluated early, this process is extremely time consuming, thus limiting the number of plants that can be analyzed. Initial attempts have been made to automate this process by developing evaluation methods for the model plant Arabidopsis thaliana. For example, Granier et al. describe a system composed of steel frame supporting trays with holes to support pots and a mechanical arm able to move according to a software program (Granier et al., 2002, New Phytologist 169: 623-635). Arabidopsis plants were grown in growth chambers and displacement sensors, a balance, a tube for irrigation and a camera were loaded onto the arm to weigh, irrigate, and take a digital picture of each pot. Boyes et al. describe a high throughput process for phenotypic analysis based on a series of defined growth stages in Arabidopsis that serve as developmental landmarks and as triggers for the collection of morphological data. Measurements were made with a caliper or ruler or by visual inspection (Boyes et al., 2001, Plant Cell 13: 1499-1510). Wang et al. describe the use of infrared thermography as a non-invasive, high throughput tool for screening of Arabidopsis guard cell signaling mutants (Wang et al., 2004, J. Exp. Bot. 55: 1187-1193).
Although high throughput screening methods have been developed for Arabidopsis, the greater size and morphological complexity of crop species limit the adaptability of these methods to breeding programs for crop species. Components of phenotyping systems adaptable to crop species have been developed. For example, U.S. Pat. No. 5,253,302 discloses a method for automatic optical classification of plants in which an image of each plant is captured by a color video camera. U.S. Published Application No. 2005/0180608 discloses a plant growth analysis system using an image acquisition system for phenotype functional analysis. The plant growth analysis system has a mechanism for conveying many observed objects which repeatedly pass a camera. U.S. Published Application No. 2007/0186313 discloses a method for the rapid evaluation of transgene function in maize plants. The method uses quantitative, non-destructive imaging technology to evaluate agronomic traits of interest in a controlled, statistically relevant greenhouse environment. U.S. Pat. No. 7,278,236 discloses an apparatus and method for nondestructively acquiring images of a plant root system. The apparatus includes a substrate for supporting the plant root system, a container for holding the substrate, an x-ray radiation source, and an x-ray image capture system. One critical aspect of plant imaging systems is the ability to turn the plant to capture images from several different angles. Systems exist that turn plants around on a fixed turntable while being photographed. Some systems provide several cameras for photographing plants at different angles. Although methods for turning plants during imaging have been developed, a need still exists for a system in which the plant or the imaging device can be turned with precision while the plant is still moved at high speed.
One of the challenges in a breeding program is to develop a system for identifying and tracking the large number of plants being evaluated. Several tracking systems which could be used for high throughput phenotype screening have been developed. U.S. Pat. No. 6,483,434 discloses a container tracking system comprising a computer system for tracking a plurality of containers or carriers. For the purpose of easily tracking any individual container or carrier, a transponder is disposed on the body of the container or carrier. U.S. Pat. No. 7,403,855 discloses an apparatus and method of tracking individual plants growing and/or taken from a growing location such as a field, growing bed, plot or greenhouse. Machine-readable data related to the individual plants is maintained in close association with the corresponding plant. U.S. Published Application No. 2004/0200146 provides an apparatus for use in conjunction with a container in which one or more plants is growing. The container has associated with it a device for receiving an enquiry signal and automatically responding by transmitting a unique identifier signal. EP1157961 discloses a container identification device which has an interrogation device with a transmitter and receiver unit using a pulse signal for interrogating identification information. This identification information is provided by a ticket attached to the container which acts as a surface wave sensor.
Another challenge in developing a system for automated analysis of plant phenotype is the need for moving large numbers of plants. Systems of moving plants for plant production have been developed. U.S. Pat. No. 5,394,646 discloses a system for automatically cultivating crops which consists of a first structure for conveying seedlings in a first predetermined path to allow seedlings to be treated under a first set of controlled growing conditions, a second structure for conveying seedlings in a second predetermined path to allow the seedlings to be treated under a second set of controlled growing conditions, and a structure for selectively diverting seedlings from the first structure onto the second structure. GB 1576010 provides an apparatus for supporting material or containers in which plants can be grown for movement along a greenhouse. The apparatus comprises at least two spaced apart parallel rails forming a track, a runner mounted on each rail for movement along the rail, and one or more elongate carrier members for supporting the material or containers. One or more carrier members are in the form of a trough extending transversely of the rails and are movable with the runners along the rails. U.S. Pat. No. 3,824,736 discloses a method for continuous plant production by moving plants on a conveyor through a corridor wherein closely controlled conditions of temperature and humidity are maintained. The corridor is formed by a series of modular units which include an illuminated section and a darkened section. Each modular unit is arranged to constitute one 24 hour growth period. U.S. Pat. No. 4,035,949 discloses an installation for rearing plants comprising a plurality of successive, independent culture chambers which include endless circulating support means on which plants are moved through zones in said chambers under controlled environmental conditions. U.S. Pat. No. 4,481,893 discloses an apparatus for use in mass growing of seedlings in a greenhouse for automatically handling seedling units comprising pots. The apparatus examines if each pot has an acceptable seedling and optionally automatically inserts a replacement seedling into any pot which needs a replacement seedling.
In determining plant phenotype, it is critical to minimize environmental variation to ensure that any differences observed among breeding lines are due to genetic variation and not simply caused by environmental effects. In conventional breeding programs, plants are grown in the field in several different locations to expose each genotype to a range of different environmental conditions. Another approach is to grow plants in controlled environments such as greenhouses and growth chambers to provide more uniform environmental conditions. However variations in several parameters, such as temperature and light intensity, often still occur. Although growth of plants in randomized complete blocks can help to mitigate the effects of environmental variation, this approach requires the growth of several plants of each genotype. Instead of minimizing environmental variation across a growing area, it is also possible to provide more uniform growth conditions by moving plants through the growing area. For example, U.S. Published Application No. 2006/0150490 discloses a process for breeding plants which comprises growing plants in an environment of controlled climatic conditions and changing the positions of the containers within the environment as required to ensure at least substantially uniform exposure of all plants in the containers to conditions in the environment.
Another aspect of evaluating plant phenotype is determination of seed quality. Seed quality measurements often require removal of the outer layers of the seed, i.e. dehulling. Development of a seed dehulling apparatus for breeding programs presents particular challenges. Existing seed dehullers work on either large amounts of seed (several kilograms) or very small amounts of seed (10-50 seeds), but breeding programs often require analysis of intermediate amounts of seed (50 to 1000 seeds) which are not easily processed by existing dehullers. Furthermore, breeding programs involve the processing of large numbers of seed lots in which each seed lot has a distinct genotype. Therefore, it is critical not to mix seed from different lots. Conventional dehullers can often trap seed in cavities of the device, allowing for contamination. Finally, since the genetic variation among seed lots can result in seeds of varying size, shape, and hull strength, the seed dehulling apparatus must be able to process a wide range of seed without causing damage. Many existing seed dehullers contain rubber rolls or concave disks with an abrasive coating (as described, for example, in U.S. Pat. No. 4,454,806) for removal of the hull. The curved surfaces of these devices can lead to breakage, especially for long, thin seeds. The seed dehullers can also be incorporated into a system for sorting and processing of seed. For example, U.S. Pat. No. 6,706,989 discloses a method and apparatus for processing seed or seed samples including an autonomous sorter which sorts seed by preprogrammed criteria. Optional features can include a counter, a cleaning device, a sheller, and a label applicator.
As mentioned above, extraction of DNA from plant tissue is often necessary in a breeding program for analysis of genotype. WO 00/63362 discloses a method for the extraction of DNA from plants. The method isolates DNA using immobilized anionic groups, preferably on a chromatographic substrate or more preferably magnetic beads derivatized with anionic groups such as diethylaminoethyl (DEAE) via an anion-exchange interaction. U.S. Pat. No. 5,523,231 discloses a method of recovering a biopolymer, including DNA, from solution involving the use of magnetically attractable beads which do not specifically bind the polymer. The beads are suspended in solution and the polymer is precipitated out of solution and becomes non-specifically associated with the beads. When the beads are magnetically drawn down the polymer is drawn down with them. The polymer can subsequently be re-dissolved and separated from the beads.
Plant breeding requires several distinct steps, including assessment of plant growth and morphology, processing of seed lots, and genetic analysis of plant tissue. Although initial advances toward automating these individual steps have been made, there is still a need to overcome the obstacles and solve problems in order to integrate these components into a highly automated, high throughput system for phenotypic and genetic analysis of crop species.